Dipeptidyl peptidases

ABSTRACT

The present invention provides an isolated peptide that comprises the sequence shown in SEQ ID NO:1 or a sequence which has at least 95% identity with SEQ ID NO:1, and which has the same substrate specificity as SEQ ID NO:1. Preferred peptides include peptides having the amino acid sequences set forth in SEQ ID NO: 3, 5, and 7. The isolated peptide is useful as a dipeptidyl aminopeptidase.

FIELD OF INVENTION

The invention relates to a dipeptidyl peptidase, to a nucleic acidmolecule which encodes it, and to uses of the peptidase.

BACKGROUND OF THE INVENTION

The dipeptidyl peptidase (DPP) IV-like gene family is a family ofmolecules which have related protein structure and function [1-3]. Thegene family includes the following molecules: DPPIV (CD26), dipeptidylamino-peptidase-like protein (DPP6) and fibroblast activation protein(FAP) [1,2,4,5]. Another possible member is DPPIV-β [6].

The molecules of the DPPIV-like gene family are serine proteases, theyare members of the peptidase family S9b, and together with prolylendopeptidase (S9a) and acylaminoacyl peptidase (S9c), they arecomprised in the prolyl oligopeptidase family[5,7].

DPPIV and FAP both have similar postproline dipeptidyl amino peptidaseactivity, however, unlike DPPIV, FAP also has gelatinase activityt[8,9].

DPPIV substrates include chemokines such as RANTES, eotaxin,macrophage-derived chemokine and stromal-cell-derived factor 1; growthfactors such as glucagon and glucagon-like peptides 1 and 2;neuropeptides including neuropeptide Y and substance P; and vasoactivepeptides[10-12].

DPPIV and FAP also have non-catalytic activity; DPPIV binds adenosinedeaminase, and FAP binds to α₃β₁ and α₅β₁ integrin[13-14].

In view of the above activities, the DPPIV-like family members arelikely to have roles in intestinal and renal handling of prolinecontaining peptides, cell adhesion, peptide metabolism, includingmetabolism of cytokines, neuropeptides, growth factors and chemokines,and immunological processes, specifically T cell stimulation[3,11,12].

Consequently, the DPPIV-like family members are likely to be involved inthe pathology of disease, including for example, tumour growth andbiology, type II diabetes, cirrhosis, autoimmunity, graft rejection andHIV infection[3,15-18].

Inhibitors of DPPIV have been shown to suppress arthritis, and toprolong cardiac allograft survival in animal models in vivo[9,20]. SomeDPPIV inhibitors are reported to inhibit HIV infection[21]. It isanticipated that DPPIV inhibitors will be useful in other therapeuticapplications including treating diarrhoea, growth hormone deficiency,lowering glucose levels in non insulin dependent diabetes mellitus andother disorders involving glucose intolerance, enhancing mucosalregeneration and as immunosuppressants[3,21-24].

There is a need to identify members of the DPPIV-like gene family asthis will allow the identification of inhibitor(s) with specificity forparticular family member(s), which can then be administered for thepurpose of treatment of disease. Alternatively, the identified membermay of itself be useful for the treatment of disease.

SUMMARY OF THE INVENTION

The present invention seeks to address the above identified need and ina first aspect provides a peptide which comprises the amino acidsequence shown in SEQ ID NO:1.

This peptide has substrate specificity for the following compounds:H-Ala-Pro-pNA, H-Gly-Pro-pNA and H-Arg-Pro-pNA. Therefore, it is aprolyl oligopeptidase and a dipeptidyl peptidase, because it is capableof hydrolysing the peptide bond C-terminal to proline in each of thesecompounds.

The peptide is homologous with human DPPIV, and importantly, identitybetween the sequences of DPPIV and SEQ ID NO: 1 is observed at theregion of DPPIV containing the catalytic triad residues and the twoglutamate residues of the β-propeller domain essential for DPPIV enzymeactivity. The observation of amino acid sequence homology means that thepeptide which has the amino acid sequence shown in SEQ ID NO:1 is amember of the DPPIV-like gene family. Accordingly the peptide wasprovisionally named DPPIVL1, and is now named and described herein asDPP8.

The following sequences of the human DPPIV amino acid sequence areimportant for the catalytic activity of DPPIV: (i)Tyr⁶²⁷GlyTrpSerTyrGlyGlyTyrVal (SEQ ID NO: 9); (ii)Ala⁷⁰⁷AspAspAsnValHisPhe (SEQ ID NO: 10); (iii) Glu⁷³⁸AspHisGlyIleAlaGln(SEQ ID NO: 11): and (iv) Tyr²⁰¹ValTyrGluGluGluVal (SEQ ID NO: 12)[25-28]. As described herein, the alignment of the following sequencesof DPP8: His⁷³⁶GlyTrpSerTyrGlyGlyTyrLeu (SEQ ID NO: 13);Leu⁸¹⁶AspGluAsnValHisPheAla (SEQ ID NO: 14); Glu⁸⁴⁷ArgHisSerIleArg (SEQID NO: 15) and Phe²⁵⁵ValLeuGlnGluGluPhe (SEQ ID NO: 16) with sequences(i) to (iv) above, respectively, suggests that these sequences of DPP8are likely to confer the catalytic activity of DPP8. Thus, in a secondaspect, the invention provides a peptide comprising the following aminoacid sequences: His⁷³⁶GlyTrpSerTyrGlyGlyTyrLeu (SEQ ID NO: 13);Leu⁸¹⁶AspGluAsnValllisPheAlaHis (SEQ ID NO: 17); Glu⁸⁴⁷ArgHisSerlleArg(SEQ ID NO: 15) and Phe²⁵⁵ValLeuGlnGluGluPhe (SEQ ID NO: 16); which hasthe substrate specificity of the sequence shown in SEQ ID NO:1.

Also described herein, using nmultiple sequence aligniment, it isobserved that DPP8 has 55% amino acid similarity and 32% amino acididentity with a C. elegans protein. Further, as shown herein, a nucleicacid molecule which encodes DPP8, is capable of hybridising specificallywith DPP8 sequeuces derived from non-human species. Together these datasuggest that DPP8 is expressed in non-human species. Thus in a thirdaspect, the invention provides a peptide which has at least 60% aminoacid identity with the amino acid sequence shown in SEQ ID NO: 1, andwhich has the substrate specificity of the sequence shown in SEQ IDNO: 1. Preferably, the amino acid identity is 75%. More preferably, theamino acid identity is 95%. Amino acid identity is calculated using GAPsoftware [GCG Version 8, Genetics Computer Group, Madison, Wis., USA] asdescribed further herein. Typically, the non-human DPP8 comprises thefollowing sequences: His⁷³⁶GlyTrpSerTyrGlyGlyTyrLeu (SEQ ID NO: 13);Leu⁸¹⁶AspGluAsnValHisPheAlaHis (SEQ ID NO: 17); Glu⁸⁴⁷ArgHisSerIleArg(SEQ ID NO: 15) and Phe²⁵⁵ValleuGlnGluGluPhe (SEQ ID NO i6).

In view of the homology between DPPIV and DPP8 amino acid sequences, itis expected that these sequences will have similar tertiary structure.This means that the tertiary structure of DPP8 is likely to include theseven-blade β-propeller domain and the α/β hydrolasc domain of DPPIV.These structures in DPP8 are likely to be conferred by the regionscomprising β-propeller, Gly¹⁸⁰ to Asp⁶⁰⁶, a/b hydrolase, Ser⁶⁰⁷ toIle⁸⁸² and about 70 to 100 residues in the region Arg³⁹ to Gln¹⁷⁹. As itis known that the β-propeller domain regulates proteolysis mediated bythe catalytic triad in the α/β hydrolase domain of Prolyloligopeptidase, [29] it is expectcd that truncated forms of DPP8 can beproduced, which have the substrate specificity sequence shown in SEQ IDNO: 1 comprising the regions referred to above(His⁷³⁶GlyTrpSerTyrGlyGlyTyrLeu (SEQ ID NO: 13):Leu⁸¹⁶AspGluAsnValHisPheAlaHis (SEQ ID NO: 17); Glu⁸⁴⁷ArgHisSerIleArg(SEQ ID NO: 15) and Phe²⁵⁵ValLeuGlnGluGluPhe (SEQ ID NO: 16)) whichconfer the catalytic specificity of DPP8. Examples of truncated forms ofDPP8 which might be prepared are those in which the region conferringthe β-propeller domain and the α/β hydrolase domain are splicedtogether. Other examples of truncated forms include those which areencoded by splice variants of DPP8 mRNA. Thus although, as describedherein, the biochemical characterisation of DPP8 shows that DPP8consists of 882 amino acids and has a molecular weight of about 100 kDa,it is recognised that truncated forms of DPP8 which have the substratespecificity of the sequence shown in SEQ ID NO: 1, may be prepared usingstandard techniques [30,31]. Thus in a fourth aspect, the inventionprovides a fragment of the sequence shown in SEQ ID NO: 1, which has thesubstrate specificity of the sequence shown in SEQ ID NO: 1. Preferably,the fragment has an amino acid sequence shown in SEQ ID NO: 3, 5 or 7.

As described herein, the sequence shown in SEQ ID NO:1 does not containa consensus sequence for N-linked glycosylation. Therefore it isunlikely that DPP8 is associated with N-linked glycosylation. In thisregard, DPP8 is distinguished from other DPPIV-like gene family members,which contain between 6 and 9 consensus sequences for N-linkedglycosylation. Thus in one embodiment, an asparagine residue in thepeptide of the first aspect of the invention is not linked to acarbohydrate molecule. The analysis of DPP8 expression described hereinshows that it is likely that DPP8 is expressed as a cytoplasmic protein.The expression of DPP8 is therefore distinguished from other DPPIV-likegene family members, which are expressed on the cytoplasmic membrane, orin other words, the cell surface membrane. Thus in another embodiment,the peptide of the first aspect of the invention is not expressed on acell surface membrane of a cell.

It is recognised that DPP8 may be fused, or in other words, linked to afurther amino acid sequence, to form a fusion protein which has thesubstrate specificity of the sequence shown in SEQ ID NO:1. An exampleof a fusion protein is described herein which comprises the sequenceshown in SEQ ID NO:1 which is linked to a further amino acid sequence: a“tag” sequence which consists of an amino acid sequence encoding the V5epitope and a His tag. An example of another further amino acid sequencewhich may be linked with DPP8 is a glutathione S transferase (GST)domain [30]. Another example of a further amino acid sequence is aportion of CD8α [8]. Thus in one aspect, the invention provides a fusionprotein comprising the amino acid sequence shown in SEQ ID NO:1 linkedwith a further amino acid sequence, the fusion protein having thesubstrate specificity of the sequence shown in SEQ ID NO:1.

It is also recognised that the peptide of the first aspect of theinvention may be comprised in a polypeptide, so that the polypeptide hasthe substrate specificity of DPP8. The polypeptide may be useful, forexample, for altering the protease susceptibility of DPP8, when used inin vivo applications. An example of a polypeptide which may be useful inthis regard, is albumin. Thus in another embodiment, the peptide of thefirst aspect is comprised in a polypeptide which has the substratespecificity of DPP8.

As described above, the isolation and characterisation of DPP8 isnecessary for identifying inhibitors of DPP8 catalytic activity, whichmay be useful for the treatment of disease. A method for identifyinginhibitors of DPP8 catalytic activity, described herein, has identifiedthat various inhibitors of DPPIV and serine proteases, zinc and mimeticpeptides, Ala-Pro-Gly and Lys-Pro, but not inhibitors ofmetalloproteinases, aspartyl proteinases or cysteinyl proteinases,inhibit DPP8 catalytic activity. Accordingly, in a fifth aspect, theinvention provides a method of identifying a molecule capable ofinhibiting cleavage of a substrate by DPP8, the method comprising thefollowing steps:

-   -   (a) contacting DPP8 with the molecule;    -   (b) contacting DPP8 of step (a) with a substrate capable of        being cleaved by DPP8, in conditions sufficient for cleavage of        the substrate by DPP8; and    -   (c) detecting substrate not cleaved by DPP8, to identify that        the molecule is capable of inhibiting cleavage of the substrate        by DPP8.

It is recognised that although inhibitors of DPP8 may also inhibit DPPIVand other serine proteases, as described herein, the alignment of theDPP8 amino acid sequence with most closely related molecules,(i.e.DPPIV), reveals that the DPP8 amino acid is distinctive, particularly atthe regions controlling substrate specificity. Accordingly, it isexpected that it will be possible to identify inhibitors which inhibitDPP8 catalytic activity specifically, which do not inhibit catalyticactivity of DPPIV-like gene family members, or other serine proteases.Thus, in a sixth aspect, the invention provides a method of identifyinga molecule capable of inhibiting specifically, the cleavage of asubstrate by DPP8, the method comprising the following steps:

-   -   (a) contacting DPP8 and a further protease with the molecule;    -   (b) contacting DPP8 and the further protease of step (a) with a        substrate capable of being cleaved by DPP8 and the further        protease, in conditions sufficient for cleavage of the substrate        by DPP8 and the further protease; and    -   (c) detecting substrate not cleaved by DPP8, but cleaved by the        further protease, to identify that the molecule is capable of        inhibiting specifically, the cleavage of the substrate by DPP8.

In a seventh aspect, the invention provides a method of reducing orinhibiting the catalytic activity of DPP8, the method comprising thestep of contacting DPP8 with an inhibitor of DPP8 catalytic activity. Asvarious inhibitors of DPPIV catalytic activity are shown herein toinhibit DPP8 catalytic activity, it is recognised that other inhibitorsof DPPIV may be useful for inhibiting DPP8 catalytic activity. Examplesof inhibitors suitable for use in the seventh aspect are described in[21,32,33]. Other inhibitors useful for inhibiting DPP8 catalyticactivity can be identified by the methods of the fifth or sixth aspectsof the invention, which methods are exemplified herein.

In one embodiment, the catalytic activity of DPP8 is reduced orinhibited in a mammal by administering the inhibitor of DPP8 catalyticactivity to the mammal. It is recognised that these inhibitors have beenused to reduce or inhibit DPPIV catalytic activity in vivo, andtherefore, may also be used for inhibiting DPP8 catalytic activity invivo. Examples of inhibitors useful for this purpose are disclosed inthe following [21,32-34].

Preferably, the catalytic activity of DPP8 in a mammal is reduced orinhibited in the mammal, for the purpose of treating a disease in themammal. Diseases which are likely to be treated by an inhibitor of DPP8catalytic activity are those in which DPPIV-like gene family members areassociated [3,10,11,17,21,36], including for example, neoplasia, type IIdiabetes, cirrhosis, autoimmunity, graft rejection and HIV infection.

Preferably, the inhibitor for use in the seventh aspect of the inventionis one which inhibits the cleavage of a peptide bond C-terminal adjacentto proline. As described herein, examples of these inhibitors are4-(2-aminoethyl)benzenesulfonylfluoride, aprotinin, benzamidine/HCl,Ala-Pro-Gly, H-Lys-Pro-OH HCl salt and zinc ions, for example, zincsulfate or zinc chloride. More preferably, the inhibitor is one whichspecifically inhibits DPP8 catalytic activity, and which does notinhibit the catalytic activity of other serine proteases, including, forexample DPPIV or FAP.

In an eighth aspect, the invention provides a method of cleaving asubstrate which comprises contacting the substrate with DPP8 inconditions sufficient for cleavage of the substrate by DPP8, to cleavethe substrate. Examples of molecules which can be cleaved by the methodare H-Ala-Pro-pNA, H-Gly-Pro-pNA and H-Arg-Pro-pNA. The conditionssufficient for cleaving the substrate are described herein. Moleculeswhich are cleaved by DPPIV including RANTES, eotaxin, macrophage-derivedchemokine, stromal-cell-derived factor 1, glucagon and glucagon-likepeptides 1 and 2, neuropeptide Y, substance P and vasoactive peptide arealso likely to be cleaved by DPP8 [11,12]. In one embodiment, thesubstrate is cleaved by cleaving a peptide bond C-terminal adjacent toproline in the substrate. The molecules cleaved by DPP8 may have Ala, orTrp, Ser, Gly, Val or Leu in the P1 position, in place of Pro [11,12].

As described herein, DPP8 gene expression is upregulated in stimulatedlymphocyte and lymphocytic cell lines which suggests that DPP8 may havea functional role in T cell costimulation and proliferation. It isrecognised therefore that measuring DPP8 gene expression is useful fordetecting T cell activation. Thus in a ninth aspect, the inventionprovides a method of detecting an activated T cell, the methodcomprising the step of detecting the level of DPP8 gene expression in aT cell. In one embodiment, the level of DPP8 gene expression is detectedby measuring the amount of DPP8 mRNA in the cell, as described herein.

The inventors have characterised the sequence of a nucleic acid moleculewhich encodes the amino acid sequence shown in SEQ ID NO:1. Thus in atenth aspect, the invention provides a nucleic acid molecule whichencodes the amino acid sequence shown in SEQ ID NO:1.

In an eleventh aspect, the invention provides a nucleic acid moleculewhich consists of the sequence shown in SEQ ID NO:2.

As described herein, at least three splice variants of DPP8 RNA whichhave an open reading frame from 2.6 to 3.1 kb in length are observed. Asa frame shift mutation or termination signal was not observed in thesequence of these splice variants, and as the coding sequence of two ofthe splice variants includes a sequence which encodes the amino acidsequence associated with catalytic activity, it is recognised that someof the peptides encoded by the splice variants are likely to have thesubstrate specificity of DPP8. Thus in an embodiment, the nucleic acidmolecule is a fragment of the sequence shown in SEQ ID NO: 1 which isabout 2.6 to 3.1 kb in length and which encodes a peptide which has thesubstrate specificity of the sequence shown in SEQ ID NO:1. Preferably,the nucleic acid molecule has a sequence shown in any one of SEQ IDNO.s: 4, 6 and 8.

In a twelfth aspect, the invention provides a nucleic acid moleculewhich is capable of hybridising to a nucleic acid molecule consisting ofthe sequence shown in SEQ ID NO:2 in stringent conditions, and whichencodes a peptide which has the substrate specificity of the sequenceshown in SEQ ID NO:1. As shown in the Northern blot analysis describedherein, DPP8 mRNA hybridises specifically to the sequence shown in SEQID NO:2, after washing in 2×SSC/1.0%SDS at 37° C., or after washing in0.1×SSC/0.1% SDS at 50° C. “Stringent conditions” are conditions inwhich the nucleic acid molecule is exposed to 2×SSC/1.0% SDS.Preferably, the nucleic acid molecule is capable of hybridising to amolecule consisting of the sequence shown in SEQ ID NO:2 in highstringent conditions. “High stringent conditions” are conditions inwhich the nucleic acid molecule is exposed to 0.1×SSC/0.1%SDS at 50° C.

As described herein, the inventors believe that the gene which encodesDPP8 is located at band q22 on human chromosome 15. The location of theDPP8 gene is distinguished from genes encoding other prolyloligopeptidases, which are located on chromosome 2, at bands 2q24.3 and2q23, or chromosome 7. Thus in an embodiment, the nucleic acid moleculeis one capable of hybridising to a gene which is located at band q22 onhuman chromosome 15.

It is recognised that a nucleic acid molecule which encodes the aminoacid sequence shown in SEQ ID NO:1, or which comprises has the sequenceshown in SEQ ID NO:2, could be made by producing the fragment of thesequence which is translated, using standard techniques [30,31]. Thus inan embodiment, the nucleic acid molecule does not contain 5′ or 3′untranslated sequences.

In a thirteenth aspect, the invention provides a vector which comprisesa nucleic acid molecule of the tenth aspect of the invention. In oneembodiment, the vector is capable of replication in a COS-7 cell, CHOcell or 293T cell, or E.coli. In another embodiment, the vector isselected from the group consisting of λTripleEx, pTripleEx, pGEM-T EasyVector, pSecTag2Hygro, pet15b, pEE14. HCMV.gs and pCDNA3.1/V5/His.

In a fourteenth aspect, the invention provides a cell which comprises avector of the thirteenth aspect of the invention. In one embodiment, thecell is an E. coli cell. Preferably, the E. coli is MC1061, DH5α, JM109,BL21DE3, pLysS. In another embodiment, the cell is a COS-7, COS-1, 293Tor CHO cell.

In a fifteenth aspect, the invention provides a method for making apeptide of the first aspect of the invention comprising, maintaining acell according to the fourteenth aspect of the invention in conditionssufficient for expression of the peptide by the cell. The conditionssufficient for expression are described herein. In one embodiment, themethod comprises the further step of isolating the peptide.

In a sixteenth aspect, the invention provides a peptide when produced bythe method of the fifteenth aspect.

In a seventeenth aspect, the invention provides a composition comprisinga peptide of the first aspect and a pharmaceutically acceptable carrier.

In an eighteenth aspect, the invention provides an antibody which iscapable of binding a peptide according to the first aspect of theinvention. The antibody can be prepared by immunising a subject withpurified DPP8 or a fragment thereof according to standard techniques[35]. As described herein, an antibody was prepared by immunising withtransiently transfected DPP8⁺ cells. It is recognised that the antibodyis useful for inhibiting activity of DPP8, or for detecting increasedgene expression of DPP8, for the purpose of identifying an activated Tcell. In one embodiment, the antibody of the eighth aspect of theinvention is produced by a hybridoma cell.

In a nineteenth aspect, the invention provides a hybridoma cell whichsecretes an antibody of the nineteenth aspect.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Cloning strategy for isolating full-length DPP8 cDNA and thealternative splicing variants of DPP8 observed. Representation of threesplice variants is shown including loss of serine recognition site byone splice variant (T8).

FIG. 2. Nucleotide sequence and amino acid sequence of human DPP8. Thenucleotide and predicted one letter code amino acid sequence are shown.This sequence shows no putative membrane spanning domain (deduced fromhydrophobicity plots) or potential N-linked glycosylation sites. Theputative serine recognition site and aspartic acid and histidine whichform the Ser-Asp-His catalytic triad are marked. Base pairs are numberedin the right margin.

FIG. 3. Alignment of the deduced amino acid residue sequence of DPP8with the C. elegans homolog of DPP8 and human DPPIV. Amino-acid residuesare numbered in the right margin. Amino-acid residues identical in allthree proteins are boxed. Asterisks mark the putative catalytic triadresidues and two glutamates of the β-propeller domain essential forDPPIV enzyme activity. The grey shading denotes the α/β hydrolase domainof these proteins. Filled triangles joined by lines indicate starts andends of alternatively spliced transcripts, stPBMCdy3-3-10 (solid lines),T8(dashed lines) and T21 (solid lines). The alignment was constructedusing the PILEUP program in GCG.

FIG. 4. Northern Blot analysis of DPP8 expression. Human multiple tissueNorthern blots (CLONTECH) containing 2 μg per lane of poly A⁺ RNA werehybridized with a ³²p labeled DPP8 probe at 68° C. and washed at highstringency. The autoradiograph was exposed for 1 day at −70° C. with aBIOMAX MS screen. Molecular mass markers are indicated in base pairs onthe left side of each autoradiogram. FIG. 4 a. Master RNA (CLONTECH)blot of poly A⁺ RNA was hybridized with a ³²p labelled DPP8 probe at 65°C. and washed at high stringency. The autoradiograph was exposed for 3days at −70° C. with BIOMAX MS screen. DPP8 mRNA was detected in alltissues examined.

FIG. 5. Chromosomal localization of human DPP8. Metaphase showing FISHwith the biotinylated DPP8 cDNA probe. Normal male chromosomes stainedwith DAPI. Hybridization sites on chromosome 15 are indicated by anarrow.

FIG. 6. Western blot analysis of transfected cell lines. Analysis oflysates of stable cell lines. DPP8 protein was seen in DPP8 /V5/Hisstable cell lines but not in DPP4 or vector-only stable cell lines. Theelectrophoretic mobility of the protein was not altered when sampleswere boiled. The band of greater mobility was probably a breakdownproduct of intact DPP8.

FIG. 7. DPP8 enzyme activity. (A) pH-dependence of DPP8 enzyme activity.(B) DPP8 and DPPIV enzyme kinetics. Means +/− SD of absorbance changeper minute, multiplied by 1000 are shown. Curve fitting assumedMichaelis-Menten kinetics.

FIG. 8. RT-PCR analysis of DPP8 expression. PCR amplifications withprimers specific for either a portion of human DPP8 that contained noalternate splicing, Val416 to Gly 679 (top of each gel) orglyceraldehyde-3-phosphate dehydrogenase (G3PDH) (bottom of each gel.(A) Top gel, lanes 1-5 contain PCR products from unstimulated PBMC cDNAfrom five subjects. Bottom gel, lanes 6 to 11 contain PCR products fromOKT3-stimulated PBMC cDNA from six subjects. (B). PCR products are fromcDNA from lymphocytic cell lines, liver or placenta as indicated.Negative control amplifications contained reaction mix, enzyme and nocDNA template. Each PCR was performed for 35 cycles. The PCR productswere electrophoresed on agarose gels and stained with ethidium bromide.The left lane of each gel contains PUC19 digested with HaeIII as sizemarkers.

FIG. 9. Northern blot analysis of murine DPP8 expression. A murineNorthern blot containing 10 μg per lane of total RNA was hybridized witha ³²P-labeled human DPP8 probe at 60° C. and washed at low stringency.Autoradiographic exposure was for 3 days at −70° C. with a BIOMAX MSscreen.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLES

General

Restriction enzymes and other enzymes used in cloning were obtained fromBoehringer Mannheim Roche. Standard molecular biology techniques wereused [31] unless indicated otherwise.

An EST clone (GENBANK™ accession number AA417787) was obtained fromAmerican Type Culture Collection. The DNA insert of this clone wassequenced on both strands using automated sequencing at SUPAMAC (Sydney,Australia).

Cell Culture and RNA Preparation

Human peripheral blood monocytes (PBMCs) were isolated by Ficoll-Hypaquedensity-gradient centrifugation (Pharmacia, Uppsala, Sweden) of bloodobtained from healthy donors. The PBMCs were incubated in AIM-V medium(Life Technologies, Gaithersburg, Md., USA) supplemented with 2 mML-glutamine and were stimulated with either 1 μg.mL⁻¹phytohaemagglutinin (Wellcome) or 100 ng.mL⁻¹ OKT3 (Orthoclone, Fla.,USA) for 72 h. The human cell lines Jurkat, CCRF-CEM, Raji, Daudi andHepG2 were grown to confluence in Dulbecco's modified Eagle's medium(Trace Biosciences, NSW, Australia) supplemented with 10% fetal bovineserum and 2 mM L-glutamine.

Liver and placental RNA were prepared from snap-frozen human tissue asdescribed previously [37]. However, RNA was prepared from PBMCs and celllines using an RNAeasy kit (Qiagen, Germany).

Bioinformatics

BLAST programs [38] and all multiple sequence alignments were performedthrough the Australian National Genomic Information Service (ANGIS,Sydney, NSW, Australia). PILEUP (GCG Version 8, Genetics Computer Group,Madison, Wis., USA) was used for multiple sequence alignments ofproteins.

A BLAST search was performed on the public expressed sequence tag (EST)database using the complete human DPPIV (GenBankwm™ accession numberX60708) and FAP (accession number U09278) nucleotide sequences as querysequences. An EST clone (accession number AA417787) was obtained fromthe American Type Culture Collection. The DNA insert of this clone wassequenced on both strands using automated sequencing at SUPAMAC (Sydney,NSW, Australia). Because of its homology with DPPIV, this new gene wasnamed dipeptidyl peptidase 8 (DPP8).

DPP8 Cloning

ESTAA417787 was used to design forward (caa ata gaa att gac gat cag gtg)and reverse (tct tga agg tag tgc aaa aga tgc) DPP8 primers forpolymerase chain reaction (PCR) from ESTAA417787. The PCR conditionswere as follows: 94° C. for 5 min, followed by 35 cycles of 94° C. for 1minute, 55° C. for 30 sec and 70° C. for 1 min. This 484 bp PCR productwas gel purified, ³²P-α labelled using Megaprime Labeling Kit (AmershamPharmacia Biotec, UK) and hybridized to a Master RNA blot (CLONTECH,Palo Alto, Calif., USA) that contained poly A⁺ from 50 adult and fetaltissues immobilized in dots as per manufacturers' instructions. ThisMaster RNA blot was also probed with DPP4 for comparison of mRNA tissueexpression.

The forward and reverse DPP8 primers were used for PCR to screen a humanplacental λ STRETCH PLUS library (CLONTECH, Palo Alto, Calif., USA) forthe presence of DPP8 cDNA in the library. The library was then screenedby standard molecular biology techniques [30,31]. After primaryscreening, 23 clones were selected for secondary screening, after which22 remained positive. For the tertiary screen the clones contained inλTripleEx were converted into pTriplEx plasmids and transformed intoBM25.8 E. coli recipient bacteria. The plated bacteria were screened andit was confirmed that all 22 clones were positive. Two of these clones,T8 and T21 were selected for further study.

5′ RACE (Rapid Amplification of cDNA Ends)

A 5′ RACE Version 2.0 kit (Gibco BRL, Life technologies) was applied onactivated T cell (ATC) and placental RNA as prescribed in the kitinstructions. The T8 DNA sequence was used to design GSP 1 (TCC TTC CTTCAG CAT CAA TC) and GSP2 (CTT AAA AGT GAC TTT AGG ATT TGC TGT ACC). 5′RACE PCR products were cloned into PGEM-T Easy®Vector (Promega Co.,Madison, Wis., USA) and sequenced by primer walking.

Confirmation of Identity of RACE Product

Reverse transcriptase PCR was carried out on ATC RNA using DPP8-pr23(GGA AGA AGA TGC CAG ATC AGC TGG) and DPP8-prl9r (TCC GTG TAT CCT GTATCA TAG AAG) to span across the junction between the RACE product andthe EST and library clones. Two gel purified products ATCd3-2-1 (1603bp)and ATC3-3-10 (1077bp) were cloned into PGEM-T Easy® (Promega Co.,Madison, Wisc., USA) and sequenced.

Subcloning of DPP8 cDNA into a pcDNA3.1/V5/His Expression Vector

The ATC RACE product, the ATCd3-2-1 (1603bp) junction fragment and thelibrary clone T21 were joined together and cloned into the expressionvector pcDNA3.1/V5/His A (Invitrogen, the Netherlands) to form a DPP8cDNA of 3.1 kb with an open reading frame of 882 aa. The first constructwas made using three sequential cloning steps. Firstly, a Eco RV/Xba Ifragment of T21 (containing 3′ DPP8, stop codon and 3′ untranslatedregion on DPP8 cDNA) was ligated into the vector pcDNA3.1/V5/His A whichhad been digested with Eco RV/Xba I. An Eco RI/Eco RV fragment ofATCd3-2-1 was then added to this construct digested with Eco RI/Eco RV.Finally the RACE product was cut with Eco RI and cloned into the Eco RIsite of the previous construct to form the complete 3.1 kb DPP8 cDNA.This construct pcDNA3.1-DPP8 expressed protein with no detectable tag.In addition the stop codon in the DPP8 expression construct inpcDNA3.1/V5/His V5 was genetically altered using PCR to create aC-terminal fusion with the V5 and His tag contained in the vector. Thisconstruct was named pcDNA3.1- DPP8/V5/His. All expression constructssubcloned into pcDNA3.1/V5/His were verified by full sequence analysis.

DPP8 Gene Expression by Northern Blot

Human multiple tissue Northern blots (CLONTECH) containing 2 ug of polyA⁺ RNA were prehybridized in Express Hybridization solution (CLONTECH)for 30 min at 68° C. Both the DPP8 484 bp product and the 5′ RACE ATCproduct were radiolabeled using a Megaprime Labeling kit (AmershamPharmacia Biotech) and [³²P]dCTP (NEN Dupont). Unincorporated label wasremoved using a NICK column (Amersham Pharmacia Biotech) and thedenatured probe was incubated for 2 hrs at 68° C. in ExpressHybridization solution. Washes were performed at high stringency andblots exposed to BIOMAX MS film for overnight with a BIOMAX MS screen at−70° C.

DPP8 Gene Expression in Mice by Northern Blot

A Northern blot containing 10 ug of total liver RNA per lane was madeusing standard methods [31]. The RNA was derived from male and femalemice of two strains, C57Bl6 and Balb/c. The Northern blot wasprehybridized in Express Hybridization solution (CLONTECH, Palo Alto,USA) for 1 hr at 60° C. A 2.4 kb human DPP8 cDNA (PCR product) wasradiolabeled using the Megaprime Labeling kit (Amersham PharmaciaBiotech) and [³²P]dCTP (NEN Dupont). Unincorporated label was removedusing a NICK column (Amersham Pharmacia Biotech) and the denatured probewas incubated with the blot overnight at 60° C. in Express Hybridizationsolution. Washes were performed at low stringency (2×SSC/0.05% SDS for 1h at 37° C. followed by 0.1×SSC/0.1% SDS for 30 min at 40° C.) and blotsexposed to BIOMAX MS film for three days with a BIOMAX MS screen at −70°C.

Expression of DPP8 in Mouse Liver Using rtPCR

Mouse liver RNA was reverse transcribed using the Superscript II enzymekit (Gibco BRL, Gaithersburg, Md.) as described previously [42]. ThecDNA was diluted 1 in 4 and stored in aliquots at −70° C. PCR usingmouseDPP8-prlF (atg att acc acc cag gaa gcg) as the forward primer andmouseDPP8-pr2R (atc tcc gac atc ttg aaa gtg acc) as the reverse primerwas used to detect mouse DPP8 mRNA.

One ul of diluted cDNA was amplified in a 50 ul PCR reaction whichcontained: 0.2 mM dNTPs, 1 ul of 50×Advantage 2 Polymerase Mix(Clontech), 1×Advantage 2 PCR buffer (Clontech) and 100 ng of eachprimer. The PCR involved an initial step of 95° C. for 1 min toinactivate the TaqStart Antibody. This was followed by 35 cycles;denaturation at 95° C. for 30 sec, 68° C. for 1 min, followed by a finalstep of 68° C. for 1 min. The amplified products were analysed byelectrophoresis of 10 μl of PCR reaction on a 3:1 Nusieve gel (FMCBioproducts, Rockville, Md.) plus 0.5 μg/ml ethidium bromide in TAEbuffer (0.04M Tris acetate, 0.001 M EDTA, pH 8.0). The gel was thenSouthern Blotted using standard techniques [31]. The Southern blot washybridized at 60° C. for 2 hr with the 2.4 kb human DPP8 cDNA probeprepared as described above. Washes were performed at low stringency(2×SSC/0.05% SDS for 1 h at 37° C. followed by 0.1×SSC/0.1% SDS for 40min at 50° C.). The blot was exposed to XAR5 Kodak film for 30 min atRT.

DPP8 Expression by RT-PCR

Reverse transcriptase PCR was performed on human ATC RNA, humanplacental RNA and human liver RNA using TED primers DPP8/pr3 (GCA CTACCT TCA AGA AAA CCT TGG) and DPP8/pr20R (TAT GGT ATT GCT GGG TCT CTCAGG) to give a 293 bp product.

Transfection, Western blot, Immunocytochemistry, Cytochemistry and FlowCytometry

Monkey kidney fibroblast (COS-7) cells (American Type CultureCollection, CRL-1651) were grown and transfected as described previously[39]. For making stable cell lines, Geneticin (G418; Gibco-BRL) wasadded to the medium, beginning 24 h after transfection. COS cellextracts were prepared by sonication followed by differentialcentrifugation and neither boiled nor reduced before SDS/PAGE (10% gel)and transfer to nitrocellulose, as described previously [40,9]. Thepresence of DPP8 fused with the V5 epitope was detected using an anti-V5mAb (Invitrogen). COS cell monolayers were fixed in cold ethanol beforestaining with anti-V5 mAb [39,41,9]. Some monolayers were fixed in 4%paraformaldehyde and permeabilized with 0.1% Triton X-100 [35], thendouble-stained with wheat germ agglutinin to label Golgi apparatus andwith goat anti-mouse IgG to label DPP8, conjugated to Alexa Fluor 488and Alexa Fluor 594, respectively (Molecular Probes, Eugene, Oreg.,USA). Flow cytometry and confocal scanning microscopy using a LeicaTCS-NT confocal microscope have been described previously [39,9].

Purification of Recombinant DPP8/V5/His and DPPIV/V5/His

Cells (1×10⁷) expressing each protein were sonicated in native buffer(50 mM sodium phosphate, 300 mM NaCl), then treated with 700 U DNAse for20 min at room temperature. DPPIV is expressed at the cell surface, so1% Triton X-100 was used to solubilize DPPIV/V5/His. Insoluble materialwas removed by centrifugation. The supernatant was incubated with 1 mLTalons® Metal Affinity Resin (Clontech) following the manufacturer'sinstructions for a batch/gravity flow procedure. The resin was washedwith 50 mM sodium phosphate, containing 300 mM NaCl and 5 mM imidazole,and proteins were eluted using the same buffer containing 150 mMimidazole. Enzyme activity was used to monitor eluted fractions.

Enzyme Assays

Enzyme assays were performed as described previously [1]. Eitherclarified cell extract from 1×10⁴ sonicated COS-7 cells or purifiedprotein derived from 1×10⁵ cells was incubated with substrate in 70 μLphosphate buffer, pH 7.4, for 30 min at 37° C., except where otherwiseindicated. The specific DPPIV substrates, Gly-Pro-toluenesulfonate,H-Gly-Pro-p-nitroanilide (NA)/HCl (Sigma, St Louis, Mo., USA) andGly-Pro-7-amino-4-trifluromethylcoumarin (Calbiochem, San Diego, Calif.,USA) were tested. Other substrates tested were H-Ala-Pro-pNA/HCl,H-Arg-Pro-pNA acetate salt, H-Lys-Ala-pNA.2HCl, H-Asp-Pro-pNA,H-Ala-Ala-pNA/HCl, H-Ala-Ala-Pro-pNA/HCl, H-Ala-Ala-Phe-pNA,succinyl-Ala-Pro-pNA, H-Ala-Phe-Pro-pNA and Z-Ala-Pro-p-NA from Bachem(Switzerland). H-Ala-Pro-4-methoxyβNA/HCl, Z-Lys-Pro-4-methoxyβNAformatesalt, H-Lys-Pro-4-methoxyβNA/HCl, Z-Ala-Pro-4-methoxyβNA, H-Gly-Pro-βNAand H-His-Ser-4-methoxyβNAacetate salt (Bachem) were tested for theirability to stain unfixed transfected cells. All inhibitors were (seeTable 2) incubated with each purified enzyme in phosphate buffer, pH7.4, for is min before the addition of substrate. After the addition of1 mM H-Ala-Pro-pNA substrate for purified DPP8 and 1 mM H-Gly-Pro-pNAsubstrate for purified DPPIV, samples were incubated for 60 min at 37°C. All enzyme assays were performed in triplicate.

Chromosomal Localization of DPP8 by Fluorescence in situ Hybridization(FISH) Analysis

DPP8 was localized using two different probes, the DPP8 EST and the T8clone. The probes were nick-translated with biotin-C¹⁴-dATP andhybridized in situ at a final concentration of 10 ng/ul to metaphasesfrom two normal males. The FISH method was modified from that previouslydescribed [37] in that chromosomes were stained before analysis withboth propidium iodide (as counterstain) and DAPI (for chromosomalidentification). Images of metaphase preparations were captured by acooled CCD camera using the Cyto Vision Ultra image collection andenhancement system (Applied Imaging International Ltd). FISH signals andthe DAPI banding pattern were merged for figure preparation.

Expression of DPP8 in Human Lymphocytes and Cell Lines

RNA (1 μg) was reverse-transcribed using the Superscript II enzyme kit(Gibco-BRL) as described previously [42]. PCR using DPP8-pr18(CTGTGACGCCACTAATTATCTATG; SEQ ID NO: 18) as the forward primer andDPP8-pr26R (CCTAGAGAGGCTAGGGTATTCAAG; SEQ ID NO: 198) as the reverseprimer was used to detect full-length DPP8 mRNA. Theglyceraldehyde-3-phosphate dehydrogcnasc (G3PDH) control primer set wasG3PDH for (ACCACAGTCCATGCCATCAC; SEQ ID NO: 20) and G3PDHrev(TCCACCACCCTGTTGCTGTA; SEQ ID NO: 21) to give a 470-bp product.

cDNA (diluted 1:4; lμg) was amplified in a 25-μL PCR mixture whichcontained: 0.2 mM dNTPs, 0.125 unit Amplitaq Gold enzyme (Perkin-Elmer),1×buffer II (Perkin-Elmer), 1.5 mM MgCl₂ and 100 ng mL⁻¹ each primer.The 35-cycle PCR was performed as follows: denaturation at 94° C. for 1min, primer annealing at 55° C. for 30 s, and an extension step at 72°C. for 1 min. The amplified products were analyzed by electrophoresis of15 μL PCR mixture on a 3:1 Nusieve gel (FMC Bioproducts, Rockville, Md.,USA) plus 0.5 μg mL⁻¹ ethidium bromide in Tris/acetate/EDTA buffer (0.04M Tris/acetate, 0.001 M EDTA, pH 8.0).

Anti-peptide Antibody

Methods followed are described in Current Protocols in Immunology [35].Two peptides were chosen using the software MacVector to predictantigenicity. The two peptides were custom synthesized (Auspep,Melbourne) and conjugated to diptheria toxin (Auspep, Melbourne).Rabbits were immunized with both peptides and serum collected at timezero and after each injection (IMVS, Adelaide.

The two peptides used were:

-   PEPTIDE Name: TEDDA-N-   SEQUENCE: CTGYTERYMGHPDQNEQG-NH2 (SEQ ID NO: 22).-   This is amino acids 773 to 789, plus a Cys at the N-terminus.-   PEPTIDE Name: TEDDR-C-   SEQUENCE: GKPYDLQIYPQERHSC-NH2 (SEQ ID NO: 23).

This is amino acids 836 to 850, plus a Cys at the C-terminus.

These sequences were taken from the C-terminal portion of DPP8.

Monoclonal antibody to DPP8

Standard methods were used for antibody production [35]. Mice wereimmunized with 2×10⁷ live COS-7 (African Green Monkey Kidney) cells thathad been transiently transfected with the DPP8 cDNA in the pcDNA3vector. The final immunisation was with CHO (Chinese Hamster Ovary)cells stably transfected with DPP8 cDNA in the pEE14 vector. Spleencells were fused with a standard fusion partner, X63Ag8 myeloma cells.Hybridoma culture supernatants were tested by immunoperoxidasehistochemistry on monolayers of the DPP8-transfected CHO cell line,using untransfected CHO cells as the negative control. Hybridomas thatproduced antibody activity were cloned.

Results

Molecular Cloning and Sequence Analysis of DPP8

The insert in ATCC EST AA417787 was 795 bp in length, containing 527 bpof coding sequence, a TAA stop codon and 258 bp of 3′ noncoding sequence(FIG. 1).

The hybridization of the Master RNA blot revealed that the genecomprising ESTAA417787 has ubiquitous tissue expression, with highlevels of expression in testis and placenta. Based on this expressionpattern, a placental cDNA library was screened with a 484 bp PCR productproduced by the forward and reverse DPP8 primers. Sequence homologyanalysis revealed that only 2 of 23 clones contained 5′ sequenceadditional to the sequence of ESTAA417787. These cDNA clones weredesignated T8 and T21, and were 1669 bp and 1197 bp respectively (FIG.1). In addition, comparison of these sequences to ESTAA417787 revealedthat T8 cDNA lacked a 153 bp (51aa) region that was present in T21 cDNAand ESTAA417787. This deletion would result in the loss of the catalyticserine (GWSYGG) in T8 cDNA. Many of the other clones characterizedappeared to contain unrelated sequence which are probably intronicsequences as a result of incomplete splicing.

The 5′ RACE technique was utilized on both ATC RNA and placental RNA toobtain the 5′ end of the DPP8 gene. The RACE product obtained fromactivated T cell RNA was 0.2 kb larger than that from placental RNA butotherwise identical (FIG. 1). The first methionine within a Kozaksequence was found 214 bp from the 5′ end of the activated T cell RACEproduct. This 5′211 bp region was 70.5% GC rich and contained a numberof potential promoter and enhancer elements (Sp1, Ap1 and ETF sites) andso was deduced to be the 5′ flanking region of the DPP8 gene. In orderto confirm the identity of the 5′ RACE product as the 5′ end of DPP8,RT-PCR was carried out to span across the junction between the RACEproduct and T8 cDNA library clone. The RT-PCR on ATC RNA produced twoclones ATCd3-2-1 and ATC3-3-10 (FIG. 1). Compared to T8 and T21, bothclones had an additional insert region of 144 bp (48 aa) immediatelyadjacent to the splice site of T8. Sequence homology analysis of thisadditional insert region found a homologous region in both the C.elegans homologue and DPP4. This clearly showed that T8 and T21 libraryclones represented splice variants of DPP8. The smaller clone ATCd3-3-10was also found to represent another splice variant of DPP8 as itcontained a 516 bp deletion at the 5′ end which would result in adeletion of 175 aa.

A full-length DPP8 clone was created using the larger RACE product,ATC3-2-1 and the T21 library clone. This generated a putative DPP8 cDNAof 3.1 kb (including 5′ and 3′ untranslated regions) with an openreading frame of 882 aa for further sequence analysis and examining DPP8function. This 882 putative DPP8 protein contained no N-linkedglycosylation sites and Kyte-Doolittle hydrophobicity analyses revealedit lacked a transmembrane domain, unlike DPP4, FAP and DPP6. Thus it islikely that DPP8 is a cytoplasmic protein (FIG. 2). The predicted DPP8protein shared 51% amino acid similarity and 27% amino acid identitywith human DPP4; the C termini of these proteins exhibited the mosthomology (FIG. 3).

Tissue Distribution of DPP8 as Determined by Master RNA and NorthernBlot

A master RNA blot was probed with a 484 nt PCR product produced by theforward and reverse DDPB primers as mentioned previously. The rnRNAtissue expression of DPP8 was ubiquitous in all human adult and fetaltissues. A similar ubiquitous expression pattern was observed using DPP4cDNA as a probe (data not shown). However, by visual assessment thegreatest levels of expression using each gene specific probe were indifferent tissues. The most intense signals using the DPP8 probe were intestis followed by placenta whereas the most intense signals using theDPP4 probe were in salivary gland and prostate gland followed byplacenta (data not shown). The probes did not bind any of the negativecontrols on the blot.

Northern blot analysis was performed on mRNA derived from differenthuman tissues (FIG. 4). Two DPP8 specific probes indicated the presenceof transcripts in all tissues examined. A transcript approximately 3.0kb in size consistent with the approximate expected size of DPP8 messagewas detected only in the testis. However, two transcripts of 8.0 and 5.0kb respectively were present in testis, spleen, peripheral bloodleukocytes and ovary at high levels; in prostrate, small intestine, andcolonic mucosa at moderate levels; and in the thymus at lower levels.The Multiple tissue Northern blot was also probed with radiolabeledhuman β-actin probe and a common 2.0 kb transcript was seen in alltissues (FIG. 4).

Expression of DPP8 in Mice Determined by Northern Blot and rtPCR

The human DPP8 cDNA sequence cross-hybridized with murine derived liverRNA. The Northern blot containing total RNA from mouse liver hybridizedto a human DPP8 probe, showing that DPP8 mRNA is expressed in mouseliver (FIG. 9A). Two mRNA transcripts of murine DPP8 were present. Thisis a similar pattern to that observed for human DPP8. These transcriptsprobably represent different length 5′ and 3′ untranslated regions ofthe murine DPP8 gene. The presence of DPP8 mRNA in the mouse liver wasalso demonstrated using rt-PCR. The primers tested generated a 537 bpPCR product. A Southern blot of this product confirmed that the murineDPP8 cross-hybridizes with human DPP8 (FIG. 9B).

Expression and functional activity of DPP8

To assess the function of DPP8 protein, the full length DPP8 cDNA of 3.1kb was cloned into the Xba I site of pcDNA3.1A/V5/His expression vectorto produce two constructs. The first construct, pcDNA3.1-DPP8, expressedDPP8 protein on its own whilst the second construct, cDNA3.1-DPP8/V5/Hisexpressed a protein with the V5 epitope and His tag fused to theC-terminus of DPP8 to facilitate analysis of protein expression.Mammalian expression constructs were stably transfected into COS-7 cellsand cellular sonicates prepared. Consistent with the molecular weightpredicted from the amino acid sequence a 100 kDa monomer was detected bywestern blotting of stable DPP8/V5/His expressing cells (FIG. 6).DPP8/V5/His protein was detected in the cytoplasmic compartment but noton the surface of ethanol fixed stable DPP8/V5/His expressing COS cells,using the anti-V5 mAb.

DPP8 is a Dipeptidyl Peptidase

Sequence homology between DPPIV and DPP8 suggested functionalsimilarities, so cell lysates of DPP8-transfected cells were examinedfor proline-specific peptidase activity. DPPIV expressed in COS-7 cellswith or without the V5/His tag were positive controls, and negativecontrols included vector-only transfected COS07 cells. Extracts ofDPP8-transfected COS-7 cells hydrolyzed H-Ala-Pro-pNA and H-Arg-Pro-pNAbut not H-Gly-Pro-pNA, H-Gly-Arg-pNA, H-Gly-Pro-toluenesulfonate orH-Gly-Pro-7-amino-4-trifluoromethylcoumarin above the levels exhibitedby untransfected COS-7 cells (data not shown). The pH optimum of DPP8enzyme activity was 7.4 (FIG. 5A), similar to the pH 7.8 optimum DPPIVenzyme activity [43,44]. DPP8 exhibited little activity below pH 6.3,suggesting that it is not an enzyme of the lysosome/endosomecompartment. Of all the substrates tested on cell monolayers, onlyAla-Pro-4MβNA/HCl stained DPP8-transfected COS cells and CHO cells (datanot shown).

Both purified recombinant DPP8/V5/His and purified recombinantDPPIV/V5/His hydrolyzed H-Ala-Pro-pNA, G-Gly-Pro-pNA and H-Arg-Pro-pNA.Transfection with DPP8 possibly causes increased dipeptidase,tripeptidase and endopeptidase activities, similar to an effect of DPPIVtransfection of melanoma cells [18]. Indeed, our results showed thatDPP8 transfected COS-7 cells, but not purified recombinant DPP8,exhibited tripeptidyl peptidase activity using the substrateH-Ala-Ala-Pro-pNA and endopeptidase activity using the substrateZ-Ala-Pro-pNA (data not shown). This was investigated further, andneither of the tripeptidyl peptidase substrates H-Ala-Ala-Phe-pNA orH-Ala-Phe-Pro-pNA [45] nor the prolyl endopeptidase substratesZ-Ala-Pro-pNA or succinyl-Ala-Pro-pNA were cleaved by purified DPP8. Ourdata clearly demonstrate that DPP8 is a dipeptidyl peptidase and lackstripeptidyl peptidase or endopeptidase activities.

The nature of the catalytic mechanism of DPP8 was further investigatedusing various inhibitors. DPP8 enzyme activity was significantlyinhibited by serine proteinase inhibitors and was insensitive toinhibitors of metalloproteinases, aspartyl proteinases and cysteineproteinases. DPP8 enzyme activity was significantly inhibited by zinc,which completely inhibits DPPIV enzyme activity [46]. The peptidesAla-Pro-Gly and Lys-Pro mimic DPP8 substrates and probably competitivelyinhibited DPP8.

Chromosomal Localization of DPP8

Two probes were used for FISH analysis, ESTAA417787 and the T8 clonefrom the placental library. Seventeen metaphases from the first normalmale were examined for fluorescent signal. All of these metaphasesshowed signal on one or both chromatids of 15 at band q22 (FIG. 5).There were a total of 2 non-specific background dots observed in thesemetaphases. A similar result was obtained from the hybridization of theprobe to 15 metaphases from the second normal male (data not shown).

Analysis of DPP8 Gene Expression by RT-PCR

DPPIV is expressed by most lymphocytes and lymphocytic cell lines butupregulated on activated lymphocytes [47, 41, 48, 49]. The varioussplice variants of DPP8 might not encode functional protein, so the PCRwas designed to detect only mRNA that contained full-length sequence(FIG. 1). At 35 cycles, amplification product of the expected size (783bp) was readily observed in OKT3-stimulated PBMCs (six of six subjects;FIG. 8) but not in unstimulated PBMCs from most subjects (four of five,FIG. 8A), suggesting that more DPP8 mRNA is expressed in activated Tcells than in unstimulated PBMCs. Similar RT-PCR data were obtained fromPBMCs stimulated with phytohaemagglutinin (data not shown). In addition,DPP8 mRNA was expressed in all B and T cell lines examined and in bothliver and placenta (FIG. 8B).

Anti-peptide Antibody

The sera of two rabbits were tested by ELISA in peptide-coated wells.Both sera bound both peptides whereas the pre-immunisation serum samplesdid not exhibit specific binding. Western blots on extracts of celllines, cell lines transfected with DPP8 cDNA and activated humanlymphocytes showed that a rabbit antiserum to the two DPP8 peptidesbinds a 100 kDa band, which is the size of DPP8. (Data not shown).

TABLE 1 K_(m) and V_(max) values for DPP8 and DPPIV K_(m) (mM) V_(max)(ΔA min⁻¹ × 1000) DPPIV DPP8 DPPIV DPP8 H-Ala- 0.374 ± 0.134 0.991 ±0.171 9.6 ± 1.0  12.4 ± 0.9  Pro-pNA H-Gly- 0.347 ± 0.088 0.467 ± 0.0647.2 ± 0.49  3.5 ± 0.14 Pro-pNATable 2. Inhibition of the peptidase activity of DPP8 in comparison withDPPIV. Common proteinase inhibitors of various enzyme types wereincubated with the purified peptidases before assay with the substratesH-Ala-Pro-pNA on DPP8 or H-Gly-Pro-pNA on DPPIV. AEBSF,4-(2-aminoethyl)benzenesulfonylfluoride.

TABLE 2 Inhibition of the peptidase activity of DPP8 in comparison withDPPIV. Common proteinase inhibitors of various enzyme types wereincubated with the purified peptidases before assay with the substratesH-Ala-Pro-pNA on DPP8 or H-Gly-Pro-pNA on DPPIV. AEBSF, 4-(2-aminoethyl)benzenesulfonylfluoride. Residual activity (% of control)Type of inhibitor Concentration DPP8 DPPIV None 100 100 Serineproteinase AEBSF  4 mM 40 52 Aprotinin  4 μg mL⁻¹ 47 81 Benzamidine/HCl10 mM 82 89 Peptides Gly-Gly-Gly 10 mM 99 106 Ala-Pro-Gly 10 mM 51 67H-Lys-Pro-OH HCl salt  4 mM 63 45 Zinc sulphate  2 mM 25 0Metalloproteinase EDTA  2 mM 115 99 Aspartate(acidic) proteinasePepstatin  2 μg mL⁻¹ 107 110 Leupeptin  0.1 mM 93 104 Cysteine(thiol)proteinase Iodoacetamide  2 mM 100 115 Dithiothreitol  2 mM 108 109Discussion

We describe the cloning, recombinant expression, biochemistry and tissueexpression of a novel human DPPIV-related postproline peptidase that wehave named DPP8. DPP8 exhibited dipeptidyl aminopeptidase but nottripeptidyl peptidase or endopeptidase activity. Like DPPIV, DPP8 wasfound to exhibit significant mRNA expression in activated T cells. Clearindications that DPP8 is a monomeric, nonglycosylated, soluble,cytoplasmic protein, which are characteristics of PEP but not of DPPIV,FAP or DPP6, were provided by our sequence and localisation data. DPP8enzyme activity had a neutral pH optimum, suggesting that it is notactive in the acidic lysosome/endosome compartment.

By homology with DPPIV, DPP8 is a member of the DPPIV-like gene family,a member of the prolyl oligopeptidase family S9b, and a member of theenzyme clan SC. The residues in DPP8 that potentially form thecharge-relay system are Ser739, Asp817 and His849 (FIG. 2). Thedipeptidyl peptidase activity of DPP8 and the absence of detectabletripeptidyl peptidase or endopeptidase activities by purified DPP8further support its placement in the S9b family. Furthermore, the DPP8substrate specificity was indistinguishable from that of thestructurally related peptidases DPPIV and FAP.

The role of DPPIV in human lymphocytes has been studied in detail usingenzyme inhibitors [49, 50-54]. DPPIV-specific inhibitors suppress bothDNA synthesis and cytokine production in vitro [48, 49, 52]. Inaddition, DPPIV-specific inhibitors decrease phorbol myristateacetate-induced tyrosine phosphorylation in human lymphocytes, furthersuggesting a role for DPPIV enzyme activity in lymphocyte activation[54]. In vivo, inhibitors of DPPIV suppress arthritis [20] and prolongcardiac allograft survival in animal models [55]. The ability of DPP8 tocleave DPPIV substrates indicates that DPPIV inhibitors may also inhibitDPP8 and that inhibitor studies may require further interpretation.Indeed, DPP8 may be responsible for some of the physiological functionsthat have been assigned to DPPIV.

FAP and DPPIV are integral membrane glycoproteins and requiredimerization for catalytic activity [19, 56, 57]. In contrast, DPP8 andPEP are non-glycosylated cytosolic proteins that are catalyticallyactive as monomers [58] and cleave Pro-Xaa bonds [43,59]. However, thesubstrate specificity of PEP is distinct from DPP8. PEP is anendopeptidase that does not cleave if a free α-amine lies N-terminal tothe proline (e.g. it does not cleave H-Ala-Pro). Recently we haveproposed that the tertiary structure of DPPIV is similar to that of PEPin having a seven-blade β-propeller domain and an α/β-hydrolase domain[3, 39, 1]. The significant sequence identity between DPP8 and DPPIVindicates that the tertiary structures of DPP8 and DPPIV are similar.However, DPP8 contains 110 amino acids more than DPPIV, so it could havean additional element of tertiary structure such as an eighth propellerblade.

The ancestral relationships between DPP8, DPPIV and FAP are reflected intheir chromosomal localization. While DPPIV and FAP have both beenlocalized to the long arm of chromosome 2, 2q24.3 [601] and 2q23 [61]respectively, DPP8 was localized to 15q22. The related genes DPP6 andPEP have been localized to chromosome 7 [621] and 6q22 respectively[63].

Two human disease loci have been mapped to 15q22. These loci are anautosomal recessive deafness locus [64] and a form of Bardet-Biedlsyndrome, type 4 [65]. Two of the clinical manifestations ofBardet-Biedl syndrome are obesity and diabetes. Attractin [66] and DPPIVhave roles in obesity [67] and diabetes [22, 68, 15] respectively and astheir substrate specificities overlap with that of DPP8, it is possiblethat DPP8 may be involved in Bardet-Biedl syndrome.

DPPIV is expressed on the surface of T cells and is a costimulatorymolecule called CD26 [3]. CD26-negative cell lines have residual DPPIVenzyme activity and PBMC have non-DPPIV derived activity against Ala-Prosubstrates [69], indicating the existence of other peptidase(s) withDPPIV-like activity. DPPIV-β exhibits a peptidase activity similar toDPPIV but is a 70-80 kDa cell surface glycoprotein [70] and is thereforedistinct from DPP8.

The biological significance of the three splice variants of DPP8 that wediscovered is unknown. None of these splice variants result in a frameshift or premature protein termination (FIG. 1). Two of the splicevariants contain all the predicted catalytic triad residues and thuspotentially produce proteins with peptidase activity. Alternate spliceforms of FAP mRNA have also been observed [71, 72]. It is possible thatexpression of splice variants may be used to regulate the levels ofactive protein. DPP8 Northern blots revealed a number of differentlysized transcripts. The predicted sizes of splice variants of DPP8 rangedfrom 2.6 to 3.1 kb whereas the large transcripts seen in most tissuesexamined in the Northern blots were 8.5 kb and 5.0 kb respectively.Similarly, two other members of the DPPIV-like gene family, DPPIV andDPP6, exhibit mRNA transcripts in Northern blots that are much largerthan the cDNA size [60, 61]. We propose that the major transcripts forDPP8 mRNA and its splice variants lie within the 5 kb band while the 8.5kb transcript(9) may contain additional 5′ and 3′ untranslatedsequences. DPP8 appears to be like DPPIV in having a ubiquitous mRNAexpression pattern by Northern analysis while being upregulated inactivated T cells. The similarities between DPP8 and DPPIV suggest thatDPP8 may, like DPPIV, play a role in T cell costimulation andproliferation. The development of DPP8 specific antibodies or inhibitorswill facilitate work in this area.

In summary, we have identified and characterized a novel humandipeptidyl aminopeptidase DPP8 with structural and functionalsimilarities to DPPIV and FAP. With many diverse biological rolessuggested for DPPIV, particularly in the immune system, and the roles ofFAP in tumor growth and liver disease, it will be interesting toinvestigate the roles of this new member of the DPPIV-like gene familyin these systems. Further work in understanding this novel protein andthe elucidation of inhibitors and physiological substrates will helpidentify the specific functions of individual members of this genefamily.

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1. An isolated peptide which comprises: (a) the sequence shown in SEQ IDNO:1; or (b) a sequence which has at least 95% identity with thesequence shown in SEQ ID NO:1, and which has the same substratespecificity as SEQ ID NO:1.
 2. A fragment of the sequence shown in SEQID NO:1 which has the same substrate specificity as SFQ ID NO:
 1. 3. Afragment according to claim 2 which consisis of the sequence shown inSEQ ID NO.s: 3, 5 or
 7. 4. A peptide according to claim 1, wherein anasparaginc residue in the peptide is not linked to a carbohydratemolecule.
 5. A peptide according to claim 1, wherein the peptide is notexpressed on the cell surface membrane of a cell.
 6. A fusion proteincomprising the amino acid sequence shown in SEQ ID NO:1 linked with afurther amino acid sequence, the fusion protein having the samesubstrate specificity as SEQ ID NO:1.
 7. A fusion protein according toclaim 6 wherein the further amino acid sequence is selected from thegroup consisting of GST, V5 epitope and His tag.
 8. A compositioncomprising a peptide according to claim 1.